C. elegans has polycistronic transcription units, similar to bacterial operons, that make polycistronic pre-mRNAs. These pre-mRNAs are processed by 3' end formation and trans-splicing, which occur in close proximity. What are the mechanims involved in production of mRNAs from polygenic precursors? Genes are separated by ~100 bp and the downstream genes are trans-spliced to SL2. We use a test operon to investigate the roles of sequences in the 3' end formation site and the intercistronic region in polycistronic pre-mRNA processing. We have firmly established a mechanistic and physical connection between the 3' end formation machinery and SL2 trans-splicing. We have discovered a U-rich sequence, Ur, near the 3' end of the upstream gene, that is required for SL2 utilization. We will determine whether CstF interacts with the Ur element to facilitate SL2 trans-splicing, or if not, what does. To determine what sequences on the SL2 snRNP are needed for trans-splicing, we have created a marked SL2 RNA gene which we have shown is correctly trans-spliced to downstream genes in operons. We have shown that C. elegans embryo extracts contain a complex containing the SL2 snRNP and CstF-64. Using a variety of mutant SL2 RNA genes, we have found that this complex is crucial for SL2 identity. Using glycerol gradients and co-immunoprecipitation experiments, we will determine additional protein components of this complex. We will also do genetic and RNAi screens for mutants in proteins involved in polycistronic pre-mRNA processing using an operon in which the downstream gene is fused to the gfp gene. We have recently performed a whole genome microarray analysis that has enabled us to identify ~90% of all C. elegans operons. Our preliminary results show that only genes encoding certain types of proteins are included in operons and that proteins that function in the same process are often co-expressed in the same operon. We will continue our bioinformatic analysis of the worm operons as a way of understanding the evolution and current function of this unusual mechanism of gene expression and regulation. We will also investigate whether SL2 trans-splicing is absolutely restricted to downstream operon trans-splice sites, and if so, how. If not, we will study the special class of non-operon trans-splice site that can be trans-spliced to SL2. Finally, we will determine whether the pre-mRNA cap attracts the SL1snRNP to non-operon trans-splice sites.